A role for NPY-NPY2R signaling in albuminuric kidney disease.

Albuminuria is an independent risk factor for the progression to end-stage kidney failure, cardiovascular morbidity, and premature death. As such, discovering signaling pathways that modulate albuminuria is desirable. Here, we studied the transcriptomes of podocytes, key cells in the prevention of albuminuria, under diabetic conditions. We found that Neuropeptide Y (NPY) was significantly down-regulated in insulin-resistant vs. insulin-sensitive mouse podocytes and in human glomeruli of patients with early and late-stage diabetic nephropathy, as well as other nondiabetic glomerular diseases. This contrasts with the increased plasma and urinary levels of NPY that are observed in such conditions. Studying NPY-knockout mice, we found that NPY deficiency in vivo surprisingly reduced the level of albuminuria and podocyte injury in models of both diabetic and nondiabetic kidney disease. In vitro, podocyte NPY signaling occurred via the NPY2 receptor (NPY2R), stimulating PI3K, MAPK, and NFAT activation. Additional unbiased proteomic analysis revealed that glomerular NPY-NPY2R signaling predicted nephrotoxicity, modulated RNA processing, and inhibited cell migration. Furthermore, pharmacologically inhibiting the NPY2R in vivo significantly reduced albuminuria in adriamycin-treated glomerulosclerotic mice. Our findings suggest a pathogenic role of excessive NPY-NPY2R signaling in the glomerulus and that inhibiting NPY-NPY2R signaling in albuminuric kidney disease has therapeutic potential.

Regulation and Function of C-Type Natriuretic Peptide (CNP) in Gonadotrope-Derived Cell Lines.

C-type natriuretic peptide (CNP) is the most conserved member of the mammalian natriuretic peptide family, and is implicated in the endocrine regulation of growth, metabolism and reproduction. CNP is expressed throughout the body, but is particularly abundant in the central nervous system and anterior pituitary gland. Pituitary gonadotropes are regulated by pulsatile release of gonadotropin releasing hormone (GnRH) from the hypothalamus, to control reproductive function. GnRH and CNP reciprocally regulate their respective signalling pathways in αT3-1 gonadotrope cells, but effects of pulsatile GnRH stimulation on CNP expression has not been explored. Here, we examine the sensitivity of the natriuretic peptide system in LßT2 and αT3-1 gonadotrope cell lines to continuous and pulsatile GnRH stimulation, and investigate putative CNP target genes in gonadotropes. Multiplex RT-qPCR assays confirmed that primary mouse pituitary tissue express Nppc,Npr2 (encoding CNP and guanylyl cyclase B (GC-B), respectively) and Furin (a CNP processing enzyme), but failed to express transcripts for Nppa or Nppb (encoding ANP and BNP, respectively). Pulsatile, but not continuous, GnRH stimulation of LßT2 cells caused significant increases in Nppc and Npr2 expression within 4 h, but failed to alter natriuretic peptide gene expression in αT3-1 cells. CNP enhanced expression of cJun, Egr1, Nr5a1 and Nr0b1, within 8 h in LßT2 cells, but inhibited Nr5a1 expression in αT3-1 cells. Collectively, these data show the gonadotrope natriuretic peptide system is sensitive to pulsatile GnRH signalling, and gonadotrope transcription factors are putative CNP-target genes. Such findings represent additional mechanisms by which CNP may regulate reproductive function.

Information Transfer via Gonadotropin-Releasing Hormone Receptors to ERK and NFAT: Sensing GnRH and Sensing Dynamics.

Information theoretic approaches can be used to quantify information transfer via cell signaling networks. In this study, we do so for gonadotropin-releasing hormone (GnRH) activation of extracellular signal-regulated kinase (ERK) and nuclear factor of activated T cells (NFAT) in large numbers of individual fixed LßT2 and HeLa cells. Information transfer, measured by mutual information between GnRH and ERK or NFAT, was <1 bit (despite 3-bit system inputs). It was increased by sensing both ERK and NFAT, but the increase was <50%. In live cells, information transfer via GnRH receptors to NFAT was also <1 bit and was increased by consideration of response trajectory, but the increase was <10%. GnRH secretion is pulsatile, so we explored information gained by sensing a second pulse, developing a model of GnRH signaling to NFAT with variability introduced by allowing effectors to fluctuate. Simulations revealed that when cell-cell variability reflects rapidly fluctuating effector levels, additional information is gained by sensing two GnRH pulses, but where it is due to slowly fluctuating effectors, responses in one pulse are predictive of those in another, so little information is gained from sensing both. Wet laboratory experiments revealed that the latter scenario holds true for GnRH signaling; within the timescale of our experiments (1 to 2 hours), cell-cell variability in the NFAT pathway remains relatively constant, so trajectories are reproducible from pulse to pulse. Accordingly, joint sensing, sensing of response trajectories, and sensing of repeated pulses can all increase information transfer via GnRH receptors, but in each case the increase is small.

Deubiquitinase Usp8 regulates α-synuclein clearance and modifies its toxicity in Lewy body disease.

In Parkinson's disease, misfolded α-synuclein accumulates, often in a ubiquitinated form, in neuronal inclusions termed Lewy bodies. An important outstanding question is whether ubiquitination in Lewy bodies is directly relevant to α-synuclein trafficking or turnover and Parkinson's pathogenesis. By comparative analysis in human postmortem brains, we found that ubiquitin immunoreactivity in Lewy bodies is largely due to K63-linked ubiquitin chains and markedly reduced in the substantia nigra compared with the neocortex. The ubiquitin staining in cells with Lewy bodies inversely correlated with the content and pathological localization of the deubiquitinase Usp8. Usp8 interacted and partly colocalized with α-synuclein in endosomal membranes and, both in cells and after purification, it deubiquitinated K63-linked chains on α-synuclein. Knockdown of Usp8 in the Drosophila eye reduced α-synuclein levels and α-synuclein-induced eye toxicity. Accordingly, in human cells, Usp8 knockdown increased the lysosomal degradation of α-synuclein. In the dopaminergic neurons of the Drosophila model, unlike knockdown of other deubiquitinases, Usp8 protected from α-synuclein-induced locomotor deficits and cell loss. These findings strongly suggest that removal of K63-linked ubiquitin chains on α-synuclein by Usp8 is a critical mechanism that reduces its lysosomal degradation in dopaminergic neurons and may contribute to α-synuclein accumulation in Lewy body disease.

Information Transfer in Gonadotropin-releasing Hormone (GnRH) Signaling: EXTRACELLULAR SIGNAL-REGULATED KINASE (ERK)-MEDIATED FEEDBACK LOOPS CONTROL HORMONE SENSING.

Cell signaling pathways are noisy communication channels, and statistical measures derived from information theory can be used to quantify the information they transfer. Here we use single cell signaling measures to calculate mutual information as a measure of information transfer via gonadotropin-releasing hormone (GnRH) receptors (GnRHR) to extracellular signal-regulated kinase (ERK) or nuclear factor of activated T-cells (NFAT). This revealed mutual information values <1 bit, implying that individual GnRH-responsive cells cannot unambiguously differentiate even two equally probable input concentrations. Addressing possible mechanisms for mitigation of information loss, we focused on the ERK pathway and developed a stochastic activation model incorporating negative feedback and constitutive activity. Model simulations revealed interplay between fast (min) and slow (min-h) negative feedback loops with maximal information transfer at intermediate feedback levels. Consistent with this, experiments revealed that reducing negative feedback (by expressing catalytically inactive ERK2) and increasing negative feedback (by Egr1-driven expression of dual-specificity phosphatase 5 (DUSP5)) both reduced information transfer from GnRHR to ERK. It was also reduced by blocking protein synthesis (to prevent GnRH from increasing DUSP expression) but did not differ for different GnRHRs that do or do not undergo rapid homologous desensitization. Thus, the first statistical measures of information transfer via these receptors reveals that individual cells are unreliable sensors of GnRH concentration and that this reliability is maximal at intermediate levels of ERK-mediated negative feedback but is not influenced by receptor desensitization.

The endosomal pathway in Parkinson's disease.

Parkinson's disease is primarily a movement disorder with predilection for the nigral dopaminergic neurons and is often associated with widespread neurodegeneration and diffuse Lewy body deposition. Recent advances in molecular genetics and studies in model organisms have transformed our understanding of Parkinson's pathogenesis and suggested unifying biochemical pathways despite the clinical heterogeneity of the disease. In this review, we summarized the evidence that a number of Parkinson's associated genetic mutations or polymorphisms (LRRK2, VPS35, GBA, ATP13A2, ATP6AP2, DNAJC13/RME-8, RAB7L1, GAK) disrupt protein trafficking and degradation via the endosomal pathway and discussed how such defects could arise from or contribute to the accumulation and misfolding of α-synuclein in Lewy bodies. We propose that an age-related pathological depletion of functional endolysosomes due to neuromelanin deposition in dopaminergic neurons may increase their susceptibility to stochastic molecular defects in this pathway and we discuss how enzymes that regulate ubiquitin signaling, as exemplified by the ubiquitin ligase Nedd4, could provide the missing link between genetic and acquired defects in endosomal trafficking. This article is part of a Special Issue entitled 'Neuronal Protein'.

Pulsatile hormonal signaling to extracellular signal-regulated kinase: exploring system sensitivity to gonadotropin-releasing hormone pulse frequency and width.

Gonadotropin-releasing hormone (GnRH) is secreted in brief pulses that stimulate synthesis and secretion of pituitary gonadotropin hormones and thereby mediate control of reproduction. It acts via G-protein-coupled receptors to stimulate effectors, including ERK. Information could be encoded in GnRH pulse frequency, width, amplitude, or other features of pulse shape, but the relative importance of these features is unknown. Here we examine this using automated fluorescence microscopy and mathematical modeling, focusing on ERK signaling. The simplest scenario is one in which the system is linear, and response dynamics are relatively fast (compared with the signal dynamics). In this case integrated system output (ERK activation or ERK-driven transcription) will be roughly proportional to integrated input, but we find that this is not the case. Notably, we find that relatively slow response kinetics lead to ERK activity beyond the GnRH pulse, and this reduces sensitivity to pulse width. More generally, we show that the slowing of response kinetics through the signaling cascade creates a system that is robust to pulse width. We, therefore, show how various levels of response kinetics synergize to dictate system sensitivity to different features of pulsatile hormone input. We reveal the mathematical and biochemical basis of a dynamic GnRH signaling system that is robust to changes in pulse amplitude and width but is sensitive to changes in receptor occupancy and frequency, precisely the features that are tightly regulated and exploited to exert physiological control in vivo.

Information transfer by leaky, heterogeneous, protein kinase signaling systems.

Cells must sense extracellular signals and transfer the information contained about their environment reliably to make appropriate decisions. To perform these tasks, cells use signal transduction networks that are subject to various sources of noise. Here, we study the effects on information transfer of two particular types of noise: basal (leaky) network activity and cell-to-cell variability in the componentry of the network. Basal activity is the propensity for activation of the network output in the absence of the signal of interest. We show, using theoretical models of protein kinase signaling, that the combined effect of the two types of noise makes information transfer by such networks highly vulnerable to the loss of negative feedback. In an experimental study of ERK signaling by single cells with heterogeneous ERK expression levels, we verify our theoretical prediction: In the presence of basal network activity, negative feedback substantially increases information transfer to the nucleus by both preventing a near-flat average response curve and reducing sensitivity to variation in substrate expression levels. The interplay between basal network activity, heterogeneity in network componentry, and feedback is thus critical for the effectiveness of protein kinase signaling. Basal activity is widespread in signaling systems under physiological conditions, has phenotypic consequences, and is often raised in disease. Our results reveal an important role for negative feedback mechanisms in protecting the information transfer function of saturable, heterogeneous cell signaling systems from basal activity.

Molecular mechanisms of gonadotropin-releasing hormone signaling: integrating cyclic nucleotides into the network.

Gonadotropin-releasing hormone (GnRH) is the primary regulator of mammalian reproductive function in both males and females. It acts via G-protein coupled receptors on gonadotropes to stimulate synthesis and secretion of the gonadotropin hormones luteinizing hormone and follicle-stimulating hormone. These receptors couple primarily via G-proteins of the Gq/ll family, driving activation of phospholipases C and mediating GnRH effects on gonadotropin synthesis and secretion. There is also good evidence that GnRH causes activation of other heterotrimeric G-proteins (Gs and Gi) with consequent effects on cyclic AMP production, as well as for effects on the soluble and particulate guanylyl cyclases that generate cGMP. Here we provide an overview of these pathways. We emphasize mechanisms underpinning pulsatile hormone signaling and the possible interplay of GnRH and autocrine or paracrine regulatory mechanisms in control of cyclic nucleotide signaling.

Signaling to extracellular signal-regulated kinase from ErbB1 kinase and protein kinase C: feedback, heterogeneity, and gating.

Many extracellular signals act via the Raf/MEK/ERK cascade in which kinetics, cell-cell variability, and sensitivity of the ERK response can all influence cell fate. Here we used automated microscopy to explore the effects of ERK-mediated negative feedback on these attributes in cells expressing endogenous ERK or ERK2-GFP reporters. We studied acute rather than chronic stimulation with either epidermal growth factor (ErbB1 activation) or phorbol 12,13-dibutyrate (PKC activation). In unstimulated cells, ERK-mediated negative feedback reduced the population-average and cell-cell variability of the level of activated ppERK and increased its robustness to changes in ERK expression. In stimulated cells, negative feedback (evident between 5 min and 4 h) also reduced average levels and variability of phosphorylated ERK (ppERK) without altering the "gradedness" or sensitivity of the response. Binning cells according to total ERK expression revealed, strikingly, that maximal ppERK responses initially occur at submaximal ERK levels and that this non-monotonic relationship changes to an increasing, monotonic one within 15 min. These phenomena occur in HeLa cells and MCF7 breast cancer cells and in the presence and absence of ERK-mediated negative feedback. They were best modeled assuming distributive (rather than processive) activation. Thus, we have uncovered a novel, time-dependent change in the relationship between total ERK and ppERK levels that persists without negative feedback. This change makes acute response kinetics dependent on ERK level and provides a "gating" or control mechanism in which the interplay between stimulus duration and the distribution of ERK expression across cells could modulate the proportion of cells that respond to stimulation.

Decoding neurohormone pulse frequency by convergent signalling modules.

GnRH (gonadotropin-releasing hormone) mediates control of reproduction. It is secreted in pulses and acts via intracellular effectors to activate gene expression. Submaximal GnRH pulse frequency can elicit maximal responses, yielding bell-shaped frequency-response curves characteristic of genuine frequency decoders. GnRH frequency decoding is therapeutically important (pulsatile GnRH can drive ovulation in assisted reproduction, whereas sustained activation can treat breast and prostate cancers), but the mechanisms are unknown. In the present paper, we review recent work in this area, placing emphasis on the regulation of transcription, and showing how mathematical modelling of GnRH effects on two effectors [ERK (extracellular-signal-regulated kinase) and NFAT (nuclear factor of activated T-cells)] reveals the potential for genuine frequency decoding as an emergent feature of the GnRH signalling network, rather than an intrinsic feature of a given protein or pathway within it.

Dual specificity phosphatases 10 and 16 are positive regulators of EGF-stimulated ERK activity: indirect regulation of ERK signals by JNK/p38 selective MAPK phosphatases.

We have explored the possible role of dual specificity phosphatases (DUSPs) on acute EGF-mediated ERK signalling using high content imaging and a delayed MEK inhibition protocol to distinguish direct and indirect effects of the phosphatases on ERK activity. Using siRNAs, we were unable to find evidence that any of the MAPK phosphatases (MKPs) expressed in HeLa cells acts directly to dephosphorylate ppERK1/2 (dual phosphorylated ERKs 1 and/or 2) in the acute time-frame tested (0-14 min). Nevertheless, siRNAs against two p38/JNK MKPs (DUSPs 10 and 16) inhibited acute EGF-stimulated ERK activation. No such effect was seen for acute effects of the protein kinase C activator PDBu (phorbol 12,13 dibutyrate) on ERK activity, although effects of EGF and PDBu on ERK-dependent transcription (Egr-1 luciferase activity) were both reduced by siRNA targeting DUSPs 10 and 16. Inhibition of EGF-stimulated ERK activity by these siRNAs was reversed by pharmacological inhibition of p38 MAPK and single cell analysis revealed that the siRNAs did not influence the nuclear-cytoplasmic distribution of ppERK1/2. Thus, DUSPs 10 and 16 are positive regulators of activation, apparently acting by modulating cross-talk between the p38 and ERK pathways. A simplified mathematical model of this scenario accurately predicted the experimental data, supporting the conclusion that the major mechanism by which MKPs influence acute EGF-stimulated ERK responses is the negative regulation of p38, resulting in the positive regulation of ERK phosphorylation and activity.

Analysis of SOX2 expression in developing human testis and germ cell neoplasia.

The transcriptional regulators of pluripotency, POU5F1 (OCT4), NANOG and SOX2, are highly expressed in embryonal carcinoma (EC). In contrast to OCT4 and NANOG, SOX2 has not been demonstrated in the early human germ cell lineage or carcinoma in situ (CIS), the precursor for testicular germ cell tumours (TGCTs). Here, we have analysed SOX2 expression in CIS and overt TGCTs, as well as normal second and third trimester fetal, prepubertal and adult testes by in situ hybridisation and immunohistochemistry using three different antibodies. In contrast to earlier studies, we detected SOX2 mRNA in most CIS cells. We also detected speckled nuclear SOX2 immunoreactivity in CIS cells with one primary antibody, which was not apparent with other primary antibodies. The results demonstrate SOX2 gene expression in CIS for the first time and raise the possibility of post-transcriptional regulation, most likely sumoylation as a mechanism for limiting SOX2 action in these cells.

Deoxyribonucleic acid methylation controls cell type-specific expression of steroidogenic factor 1.

Steroidogenic factor 1 (SF1) is expressed in a time- and cell-specific manner in the endocrine system. In this study we present evidence to support that methylation of CpG sites located in the proximal promoter of the gene encoding SF1 contributes to the restricted expression pattern of this nuclear receptor. DNA methylation analyses revealed a nearly perfect correlation between the methylation status of the proximal promoter and protein expression, such that it was hypomethylated in cells that express SF1 but hypermethylated in nonexpressing cells. Moreover, in vitro methylation of this region completely repressed reporter gene activity in transfected steroidogenic cells. Bisulfite sequencing of DNA from embryonic tissue demonstrated that the proximal promoter was unmethylated in the developing testis and ovary, whereas it was hypermethylated in tissues that do not express SF1. Together these results indicate that the DNA methylation pattern is established early in the embryo and stably inherited thereafter throughout development to confine SF1 expression to the appropriate tissues. Chromatin immunoprecipitation analyses revealed that the transcriptional activator upstream stimulatory factor 2 and RNA polymerase II were specifically recruited to this DNA region in cells in which the proximal promoter is hypomethylated, providing functional support for the fact that lack of methylation corresponds to a transcriptionally active gene. In conclusion, we identified a region within the SF1/Sf1 gene that epigenetically directs cell-specific expression of SF1.

Origin of pluripotent germ cell tumours: the role of microenvironment during embryonic development.

Carcinoma in situ (CIS) testis, known also as intratubular germ cell neoplasia, is the cancer stem cell from which the great majority of testicular germ cell derived tumours (TGCTs) of the testis arise. TGCTs can proliferate into morphologically homogeneous seminomas or can differentiate into virtually any type of tissue and form teratomas (non-seminomas). CIS cells display a close phenotypic similarity to fetal germ cells (primordial germ cells or gonocytes) suggesting an origin due to a developmental delay or arrest of differentiation of early germ cells. The pluripotency of these neoplasms has recently been explained by a close resemblance of their expression profile to that of embryonic inner cell mass cells studied in culture as embryonic stem cells, with high expression of transcription factors associated with pluripotency, such as NANOG and OCT3/4, as well as proteins found in several tissue specific stem cells, such as TFAP2C (AP-2gamma) or KIT. CIS and seminomas highly express a number of pre-meiotic germ cell specific genes, which are down-regulated during development to non-seminomas, while the expression of other embryonic markers, such as SOX2, is up-regulated. The mechanistic pathways and causative factors remain to be elucidated of both the initial transformation of fetal germ cells into CIS cells and the progression of CIS cells into an invasive tumour in the young adult. However, evidence supported by epidemiological studies indicate that disturbances in the hormonal microenvironment of the differentiating gonads may results in both the neoplasia and a host of other problems later in life, such as genital malformations, decreased spermatogenesis, and signs of hypogonadism.

The early human germ cell lineage does not express SOX2 during in vivo development or upon in vitro culture.

NANOG, POU5F1, and SOX2 are required by the inner cell mass of the blastocyst and act cooperatively to maintain pluripotency in both mouse and human embryonic stem cells. Inadequacy of any one of them causes loss of the undifferentiated state. Mouse primordial germ cells (PGCs), from which pluripotent embryonic germ cells (EGCs) are derived, also express POU5F1, NANOG, and SOX2. Thus, a similar expression profile has been predicted for human PGCs. Here we show by RT-PCR, immunoblotting, and immunohistochemistry that human PGCs express POU5F1 and NANOG but not SOX2, with no evidence of redundancy within the group B family of human SOX genes. Although lacking SOX2, proliferative human germ cells can still be identified in situ during early development and are capable of culture in vitro. Surprisingly, with the exception of FGF4, many stem cell-restricted SOX2 target genes remained detected within the human SOX2-negative germ cell lineage. These studies demonstrate an unexpected difference in gene expression between human and mouse. The human PGC is the first primary cell type described to express POU5F1 and NANOG but not SOX2. The data also provide a new reference point for studies attempting to turn human stem cells into gametes by normal developmental pathways for the treatment of infertility.

Evaluating human embryonic germ cells: concord and conflict as pluripotent stem cells.

The realization of cell replacement therapy derived from human pluripotent stem cells requires full knowledge of the starting cell types as well as their differentiated progeny. Alongside embryonic stem cells, embryonic germ cells (EGCs) are an alternative source of pluripotent stem cell. Since 1998, four groups have described the derivation of human EGCs. This review analyzes the progress on derivation, culture, and differentiation, drawing comparison with other pluripotent stem cell populations.